Actin filament lengths are precisely regulated and very stable in the red blood cell (RBC) membrane skeleton, while in platelets, actin filament lengths are dynamically regulated during receptor- mediated actin assembly as platelets extend lamellipodia during thrombogenesis. The broad, long term objective of this research is to elucidate how actin filament length and turnover is controlled in RBCs and platelets. An additional objective is to determine the role of actin filament length regulation in RBC membrane skeleton biogenesis and stability, and how aberrant filament length regulation may result in abnormal RBCs and hemolytic anemias. This proposal focuses on molecular mechanisms and in vivo functions of tropomodulins (Tmods), tropomyosin (TM)-regulated actin filament pointed end-capping proteins in RBCs (Tmodl) and platelets (TmodS).
The specific aims are: (1) To investigate the molecular basis for Tmodl or TmodS binding to RBC or platelet TMs, and for TM-regulated actin capping. Biochemical and biophysical assays will be used to identify TM isoform-specific binding and TM-actin capping domains and a novel TmodS monomer-binding site. (2) To establish the in vivo function of Tmodl in regulation of RBC actin filament length, membrane skeleton assembly and stability using mouse models. Tmodl and TmodS conditional knockout mice will be generated followed by breeding with a v,LCR-ppr- Cre mouse to produce single or double knockouts for Tmodl and/or TmodS in RBCs and platelets. RBC phenotypes will be examined by hematology, ektacytometry,membrane skeleton assembly, and electron microscopy of actin filament lengths in membrane skeletons. (3) To investigate an in vivo function for TmodS in receptor-mediated actin assembly during activation and spreading of platelets. Platelet function in TmodS-deficient mouse platelets will be evaluated by hematological analyses, bleeding times, and thrombus formation under flow ex vivo. A role for TmodS in actin assembly, shape change and spreading will be evaluated by biochemical assays, immunofluorescence and electron microscopy, and by time-lapse interference reflection and fluorescence microscopy of lamellipodia and filopodia protrusior in living platelets. Molecular requirements for TmodS function will be tested using a permeabilized platelet model for receptor-coupled platelet activation, by introduction of TmodS or Tmodl domains and mutants.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL083464-24
Application #
7761293
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Sarkar, Rita
Project Start
1984-12-01
Project End
2011-06-30
Budget Start
2009-12-01
Budget End
2011-06-30
Support Year
24
Fiscal Year
2010
Total Cost
$718,156
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Gokhin, David S; Fowler, Velia M (2017) Software-based measurement of thin filament lengths: an open-source GUI for Distributed Deconvolution analysis of fluorescence images. J Microsc 265:11-20
Sui, Zhenhua; Gokhin, David S; Nowak, Roberta B et al. (2017) Stabilization of F-actin by tropomyosin isoforms regulates the morphology and mechanical behavior of red blood cells. Mol Biol Cell 28:2531-2542
Wu, Tongbin; Mu, Yongxin; Bogomolovas, Julius et al. (2017) HSPB7 is indispensable for heart development by modulating actin filament assembly. Proc Natl Acad Sci U S A 114:11956-11961
Nowak, Roberta B; Papoin, Julien; Gokhin, David S et al. (2017) Tropomodulin 1 controls erythroblast enucleation via regulation of F-actin in the enucleosome. Blood 130:1144-1155
Gokhin, David S; Fowler, Velia M (2016) Feisty filaments: actin dynamics in the red blood cell membrane skeleton. Curr Opin Hematol 23:206-14
Gokhin, David S; Ochala, Julien; Domenighetti, Andrea A et al. (2015) Tropomodulin 1 directly controls thin filament length in both wild-type and tropomodulin 4-deficient skeletal muscle. Development 142:4351-62
Fischer, Robert S; Fowler, Velia M (2015) Thematic Minireview Series: The State of the Cytoskeleton in 2015. J Biol Chem 290:17133-6
Gokhin, David S; Nowak, Roberta B; Khoory, Joseph A et al. (2015) Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane. Mol Biol Cell 26:1699-710
Sui, Zhenhua; Nowak, Roberta B; Sanada, Chad et al. (2015) Regulation of actin polymerization by tropomodulin-3 controls megakaryocyte actin organization and platelet biogenesis. Blood 126:520-30
Gokhin, David S; Tierney, Matthew T; Sui, Zhenhua et al. (2014) Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle. Mol Biol Cell 25:852-65

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